EP0077515A2 - Procédé et réactif pour la détermination de l'hémoglobine glycosylée - Google Patents

Procédé et réactif pour la détermination de l'hémoglobine glycosylée Download PDF

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Publication number
EP0077515A2
EP0077515A2 EP82109382A EP82109382A EP0077515A2 EP 0077515 A2 EP0077515 A2 EP 0077515A2 EP 82109382 A EP82109382 A EP 82109382A EP 82109382 A EP82109382 A EP 82109382A EP 0077515 A2 EP0077515 A2 EP 0077515A2
Authority
EP
European Patent Office
Prior art keywords
glycosylated
hemoglobin
haptoglobin
glycosylated hemoglobin
differentiation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP82109382A
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German (de)
English (en)
Other versions
EP0077515A3 (en
EP0077515B1 (fr
Inventor
Rolf Dr.Rer.Nat. Deeg
Urban Schmitt
Joachim Dr.Rer.Nat. Ziegenhorn
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Roche Diagnostics GmbH
Original Assignee
Roche Diagnostics GmbH
Boehringer Mannheim GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Roche Diagnostics GmbH, Boehringer Mannheim GmbH filed Critical Roche Diagnostics GmbH
Priority to AT82109382T priority Critical patent/ATE12318T1/de
Publication of EP0077515A2 publication Critical patent/EP0077515A2/fr
Publication of EP0077515A3 publication Critical patent/EP0077515A3/de
Application granted granted Critical
Publication of EP0077515B1 publication Critical patent/EP0077515B1/fr
Expired legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
    • G01N33/721—Haemoglobin
    • G01N33/723—Glycosylated haemoglobin
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/10—Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
    • Y10T436/107497—Preparation composition [e.g., lysing or precipitation, etc.]

Definitions

  • the invention relates to a method for the determination of glycosylated hemoglobin in blood samples.
  • HbA Glycosylated hemoglobin
  • HbA O non-enzymatic glycosylation of hemoglobin
  • glycosylated portion of total hemoglobin has gained importance in diabetes diagnostics in recent years (see L. Jovanovic and C.M. Peterson, Am.J. Med. 70 (1981) pp. 331-338).
  • the reaction between individual hemoglobin molecules and the glucose results in a stable reaction product that is retained throughout the entire lifespan of the erythrocytes, i.e. around 100-200 days.
  • Short-term fluctuations in the blood sugar content do not have a decisive influence on the concentration of glycosylated hemoglobin.
  • the concentration of glycosylated hemoglobin therefore reflects the average glucose concentration in the blood of a patient over a long period of time relatively accurately.
  • a method for determining the glycosylated hemoglobin in blood samples is known from German Offenlegungsschrift 29 50 457, in which the change in the spectroscopic properties of a blood sample when an allosteric effector is added is used to determine the HbA 1 . Only the non-glycosylated main part of the hemoglobin is affected. The measured extinction differences are very small in the relevant range. They also decrease as the glycosylated portion of total hemoglobin increases.
  • the object of the invention was therefore to provide a method with which the glycosylated hemoglobin can be determined reliably and precisely in a fast, technically simple manner.
  • the method according to the invention in which after a chemical or physical treatment of the blood sample to release the hemoglobin from the erythrocytes, a differentiation of the glycosylated and non-glycosylated hemoglobin portion is carried out with the help of haptoglobin and either the reaction between hemoglobin and haptoglobin is kinetic followed or the one bound to haptoglobin or the proportion of hemoglobin not bound by haptoglobin is measured by known methods.
  • Differentiation of glycosylated and non-glycosylated hemoglobin means all methods that allow a distinction between glycosylated and non-glycosylated hemoglobin based on their chemical and physical properties.
  • the present invention thus relates to a method for the determination of glycosylated hemoglobin in blood samples, glycosylated and non-glycosylated hemoglobin being released from the erythrocytes by chemical or physical treatment of the blood sample, optionally converting the hemoglobin into methemoglobin, and then differentiating between glycosylated and non-glycosylated Hemoglobin performed and then the non-glycosylated or glycosylated portion of the hemoglobin is determined in a known manner, characterized in that the differentiation of glycosylated and non-glycosylated hemoglobin is carried out with the help of haptoglobin.
  • the process according to the invention can be carried out in the presence of a suitable buffer system.
  • a suitable buffer system in the pH range from 4.0 to 8.5, preferably from 6.0 to 7.0, can be used as a suitable buffer system within the scope of the invention.
  • Phosphate and bis-tris buffers have proven particularly useful.
  • Haptoglobin is a plasma protein. It has long been known that haptoglobin is able to bind hemoglobin released from the erythrocytes (see also T. Sasazuki, Immunochemistry 8 (1971) pp. 695-704).
  • glycosylated and non-glycosylated hemoglobin differ in terms of their binding behavior differentiate against haptoglobin.
  • the glycosylated hemoglobin is bound to the haptoglobin more quickly than the non-glycosylated hemoglobin. This follows, for example, from the decrease in the fluorescence intensity of a measurement solution containing haptoglobin after the addition of glycosylated or non-glycosylated hemoglobin.
  • the measurement of the fluorescent radiation which is a measure of the interaction between hemoglobin and haptoglobin, can be carried out in a known manner. In Fig. 1 is the decrease
  • the method for determining the glycosylated portion of the total hemoglobin is expediently carried out in such a way that the glycosylated and the non-glycosylated hemoglobin are first released from the erythrocytes by customary methods.
  • a solution containing haptoglobin is added to the hemolyzed blood sample, optionally after adding a suitable oxidizing agent to convert the hemoglobin into the methemoglobin.
  • the glycosylated portion of the hemoglobin is preferably bound to the haptoglobin. Conventional measuring methods are used to distinguish the bound from the unbound portion of hemoglobin or methemoglobin.
  • inositol hexaphosphate The influence of inositol hexaphosphate on the binding behavior of glycosylated and non-glycosylated hemoglobin compared to haptoglobin is clear from FIG. 2.
  • the decrease in the fluorescence intensity of a haptoglobin-containing solution after addition of inositol hexaphosphate and non-glycosylated hemoglobin is plotted against time.
  • the course of the measurement curves clearly shows that in the presence of inositol hexaphosphate, glycosylated hemoglobin binds to haptoglobin much faster than the non-glycosylated hemoglobin.
  • hemoglobin normally present after the hemolysis of the erythrocytes into the methemoglobin before the differentiation of glycosylated and non-glycosylated hemoglobin.
  • a number of known methods are available to the person skilled in the art, for example oxidation with potassium hexacyanoferrate (III) or with sodium nitrite.
  • a reducing agent for example sodium dithionite
  • the haptoglobin is used in at least an equimolar amount based on the hemoglobin.
  • an essential upper margin is also advantageous here.
  • Preferably 2 to 50 times the molar amount is used.
  • the haptoglobin can also be covalently bound to an insoluble carrier. All customary carrier materials suitable for fixing proteins are suitable as carriers.
  • the haptoglobin is linked to the carrier material in a manner which is generally known to the person skilled in the art.
  • the carrier-fixed haptoglobin is added to the hemolized blood sample, which may have been provided with the additives mentioned above. After sufficient contact time, e.g. after about 1 minute, the insolubilized haptoglobin with the bound glycosylated hemoglobin portion is separated off and the glycosylated hemoglobin is determined in the usual way either directly photometrically on the basis of its own color or on the basis of its pseudo-peroxidative activity.
  • Another object of the invention is a reagent for the differentiation of glycosylated and non-glycosylated hemoglobin, which contains haptoglobin in free or carrier-bound form.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Hematology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)
  • Saccharide Compounds (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
EP82109382A 1981-10-16 1982-10-11 Procédé et réactif pour la détermination de l'hémoglobine glycosylée Expired EP0077515B1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AT82109382T ATE12318T1 (de) 1981-10-16 1982-10-11 Verfahren und reagenz zur bestimmung von glykosiliertem haemoglobin.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE3141146 1981-10-16
DE19813141146 DE3141146A1 (de) 1981-10-16 1981-10-16 Verfahren und reagens zur bestimmung von glykosiliertem haemoglobin

Publications (3)

Publication Number Publication Date
EP0077515A2 true EP0077515A2 (fr) 1983-04-27
EP0077515A3 EP0077515A3 (en) 1983-08-17
EP0077515B1 EP0077515B1 (fr) 1985-03-20

Family

ID=6144259

Family Applications (1)

Application Number Title Priority Date Filing Date
EP82109382A Expired EP0077515B1 (fr) 1981-10-16 1982-10-11 Procédé et réactif pour la détermination de l'hémoglobine glycosylée

Country Status (14)

Country Link
US (1) US4438204A (fr)
EP (1) EP0077515B1 (fr)
JP (1) JPS5879163A (fr)
AT (1) ATE12318T1 (fr)
AU (1) AU537363B2 (fr)
CA (1) CA1199257A (fr)
DD (1) DD203778A5 (fr)
DE (2) DE3141146A1 (fr)
DK (1) DK157047C (fr)
ES (1) ES8306406A1 (fr)
FI (1) FI73088C (fr)
HU (1) HU188211B (fr)
IE (1) IE53534B1 (fr)
ZA (1) ZA827547B (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0215401A3 (fr) * 1985-09-14 1987-05-20 Roche Diagnostics GmbH Procédé et réactif pour la détermination de l'hémoglobine glycosylée ainsi que composés appropriés et procédé pour leur préparation
DE19856433A1 (de) * 1998-12-08 2000-06-21 Centeon Pharma Gmbh Verfahren zum spezifischen Nachweis von glykosilierten Proteinen
EP2562539A4 (fr) * 2010-03-31 2015-01-28 Sekisui Medical Co Ltd Procédé d'analyse des hémoglobines

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3314308A1 (de) * 1983-04-20 1984-10-25 Boehringer Mannheim Gmbh, 6800 Mannheim Verfahren und reagens zur bestimmung des haemoglobin-haptoglobin komplexes in gegenwart von freiem haemoglobin
US4837170A (en) * 1986-01-20 1989-06-06 Otsuka Pharmaceutical Co., Ltd. Monoclonal antibody, measuring method of glucosylated protein by utilizing the monoclonal antibody, and kit therefor
EP0299419B1 (fr) * 1987-07-14 1993-10-20 Kabushiki Kaisha Kyoto Daiichi Kagaku Méthode de mesure automatique de glyco-hémoglobine
US4970171A (en) * 1987-11-09 1990-11-13 Miles Inc. Denaturant reagents for convenient determination of hemoglobin derivatives in blood
JP3032891B2 (ja) * 1989-03-04 2000-04-17 吉富製薬株式会社 遊離ヘモグロビン測定用試薬キット及びそれを用いる遊離ヘモグロビンの測定法
US5484735A (en) * 1989-08-23 1996-01-16 Northwestern University Immunoassay of glycosylated proteins employing antibody directed to reductively glycosylated N-terminal amino acids
AU3737093A (en) * 1992-03-04 1993-10-05 Abbott Laboratories Determination of glycated hemoglobin by fluorescence quenching
US5739037A (en) * 1992-09-29 1998-04-14 Drew Scientific Limited Process for eliminating labile glycohaemoglobin from a sample
EP0668786A1 (fr) * 1992-11-03 1995-08-30 Chronomed, Inc. Methodes et processus de preparation de fractions de globules rouges
US5424447A (en) * 1993-07-07 1995-06-13 The Medical College Of Wisconsin Research Foundation, Inc. Heme binding compounds and use thereof
US6174734B1 (en) * 1997-10-24 2001-01-16 Abbott Laboratories Measurement of glycated hemoglobin
DE102009053225A1 (de) * 2009-11-06 2011-05-12 Deutsche Sporthochschule Köln Verfahren zur Bestimmung der totalen Hämoglobinmenge im Körper eines Lebewesens

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3350174A (en) * 1964-01-08 1967-10-31 Miles Lab Process of determining hemoglobin separated from peroxidase inhibitors in urine
US4189304A (en) 1978-10-27 1980-02-19 Miles Laboratories, Inc. Device and method for detecting myoglobin
US4200435A (en) * 1978-12-26 1980-04-29 Abbott Laboratories Determination of glycosylated hemoglobin in blood
US4268270A (en) * 1979-04-30 1981-05-19 Children's Hospital Medical Center Glycosylated hemoglobin measurement
US4255385A (en) 1979-10-23 1981-03-10 Abbott Laboratories Reagent and test kit for determining glycosylated hemoglobin
US4371374A (en) 1980-11-17 1983-02-01 The Rockefeller University Monitoring metabolic control in diabetic patients by measuring glycosylated amino acids and peptides in urine

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0215401A3 (fr) * 1985-09-14 1987-05-20 Roche Diagnostics GmbH Procédé et réactif pour la détermination de l'hémoglobine glycosylée ainsi que composés appropriés et procédé pour leur préparation
DE19856433A1 (de) * 1998-12-08 2000-06-21 Centeon Pharma Gmbh Verfahren zum spezifischen Nachweis von glykosilierten Proteinen
DE19856433C2 (de) * 1998-12-08 2001-09-13 Aventis Behring Gmbh Verfahren zum spezifischen Nachweis von glykosilierten Proteinen
EP2562539A4 (fr) * 2010-03-31 2015-01-28 Sekisui Medical Co Ltd Procédé d'analyse des hémoglobines
US9304139B2 (en) 2010-03-31 2016-04-05 Sekisui Medical Co., Ltd. Method of analyzing hemoglobins

Also Published As

Publication number Publication date
ES516496A0 (es) 1983-06-01
DK458682A (da) 1983-04-17
DK157047B (da) 1989-10-30
EP0077515A3 (en) 1983-08-17
ES8306406A1 (es) 1983-06-01
JPS5879163A (ja) 1983-05-12
CA1199257A (fr) 1986-01-14
AU537363B2 (en) 1984-06-21
JPH0153748B2 (fr) 1989-11-15
EP0077515B1 (fr) 1985-03-20
DE3141146A1 (de) 1983-04-28
US4438204A (en) 1984-03-20
FI823454L (fi) 1983-04-17
FI73088B (fi) 1987-04-30
DK157047C (da) 1990-04-02
FI823454A0 (fi) 1982-10-11
HU188211B (en) 1986-03-28
IE822493L (en) 1983-04-16
AU8889682A (en) 1983-04-21
DE3262693D1 (en) 1985-04-25
FI73088C (fi) 1987-08-10
DD203778A5 (de) 1983-11-02
ZA827547B (en) 1983-09-28
IE53534B1 (en) 1988-12-07
ATE12318T1 (de) 1985-04-15

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